Enzyme Kinetics and Inhibition Flashcards
Describe enzyme kinetics.
- Enzyme and substrate must combine to form ES complex, then enzyme must be recycled after the reaction is finished,
- Reaction at equilibrium with formation of ES complex, with rates k 1 and k -1
- Rate limiting step is production of product, driven by k 2 rate.
- As reaction is thermodynamically stable there is very little conversion of product back to substrate, so k -2 ignored
What is the relationship between the concentration if a substrate and enzymatic rate?
- First Order: Rate dependent on [S]
- Zero Order: No relationship between V and [S]
- Second Order: Relationship between V and [S] not proportional to [S], but rather multiple substrates
To study enzymes, first order kinetics must be present:
- Velocity increases as [S] increases (first order kinetics) up to a point where the enzyme is “saturated” with substrate (Vmax)
- At Vmax, rate of the reaction unaffected by increases [S] - all enzyme active sites in use. (zero order kinetics)
Describe enzyme conc. and reaction rate.
- Rate of reaction increases as enzyme concentration increases (constant [S])
- At higher enzyme concentrations, greater availability to catalyse reaction
- Linear relationship between reaction rate and [E] (at constant [S])
What does the machetes-menten equation assume?
- Equilibrium - the association and dissociation of the substrate and enzyme
is assumed to be a rapid equilibrium.
E+S ⟶ ES is fast
ES ⟶ E+ P is rate limiting - Steady state - ES immediately comes to steady state and is a constant.
i.e. ES is formed as fast as enzyme releases the product. - At early time points, at initial velocity (V0), [P] ≈ 0
- Enzyme exists in only two forms: free (E) and substrate-bound (ES)
What is the Michaelis-menten equation?
V0 = Vmax[S]/Km+[S]
What is the Michaelis Constant (Km)
Km = k-1 + k2/k1
Describe enzymes characterised by Km
- [S] at which the rate of reaction is half its maximum (1/2 Vmax)
- Represents dissociation constant (substrate affinity) of ES.
- Low values indicate ES complex held together tightly and rarely
dissociates without S first reacting to form P.
What is the physiological relevance of Km?
Utilisation of glucose in the liver
* Glucose converted by two different kinases to form glucose-6-phosphate.
glucose + ATP > glucose 6-phosphate + ADP
KM for glucose: Hexokinase = 0.05 mM, Glucokinase = 0.5 mM.
* Low blood sugar (fasted state), hexokinase phosphorylates glucose;
* When blood glucose rises (feeding), the high KM enzyme also functions.
Describe enzyme catalytic constant kcat
- At Vmax with high [S], rate determined by [E].
- Rate constant under these conditions is catalytic constant, kcat = k2
- kcat = turnover number
= max number of S converted to P per second by each active site. - Measures how fast a given enzyme can catalyze a specific reaction
(units = s -1) - Larger k cat = more rapid catalytic events at the enzyme’s active site
What are the real world limitation of Michaelis-menten kinetics?
Michaelis-Menten kinetics relies on law of mass action: assumes free diffusion and thermodynamically-driven random collision.
Many biochemical or cellular processes deviate from these conditions:
* Cytoplasm behaves more like a gel than a freely flowable aqueous
solution, severely limiting molecular movements (diffusion or collision).
* Heterogeneous enzymatic reactions - molecular mobility of E or S can be
restricted, due to immobilisation or phase-separation of reactants.
e.g. membrane enzymes.
* Homogenous enzymatic reactions - mobility of E or S may be limited
e.g. DNA polymerase where E moves along a chained substrate,
rather than having a three-dimensional freedom.
Describe random substrate binding (Ternary Complex).
- Assumes independent binding of substrates and products
- Two independent binding sites; Substrate binding independent of other substrate
Describe the ordered substrate binding (Ternary complex)
- One substrate must bind before second substrate can bind effectively
Describe the ping-pong mechanism.
- Enzyme binds substrate A and then releases P
- Intermediate form of enzyme (E*) often carries A fragment and then binds B
- Product Q released and Enzyme returns to original state (E)
Describe competitive inhibitor.
- Reversible and has a structure similar to S
- Competes with S for active site
- Effect reversed by increasing [S]